The present invention relates to liquid chromatograph, and more particularly to a liquid chromatograph having a pre-focusing column used for condensing a specimen to be analyzed, and a method of analysis of a specimen including a condensation process.
Liquid chromatography is used extensively in the field of chemical analysis for separating and analyzing specimen quantitatively. Particularly, the liquid chromatography, which uses a semi-micro column having an inner diameter of 1-2 mm, is advantageous for providing a high sensitivity, high resolution and high precision analysis. Thus, intensive investigations are being made on the art of liquid chromatography.
In particular, there is an emphasis in the art of using a semi-micro column or a short general-purpose column (such as the one having an inner diameter of 4 mm and a length of 10 mm or 20 mm) for a pre-focusing column, not for a separation column, wherein the pre-focusing column is used for condensing the specimen.
The pre-focusing column is provided separately to the usual separation column in a liquid chromatograph, and is used prior to the separation of the specimen in the separation column. By using the pre-focusing column, impurities are removed from a sample solution and the specimen is condensed in the sample solution.
Inside the pre-focusing column, a column packing material corresponding to the specimen to be analyzed is provided, such that the sample solution is condensed by the foregoing column packing material.
Thus, the separation process in the separation column is made based on the condensed specimen solution, and the performance of separation in the separation column is improved significantly.
The use of a small-volume, semi-micro column for the purpose of pre-focusing column is particularly advantageous for improving the efficiency of condensation therein and hence for improving the performance of separation in the separation column.
On the other hand, a specimen to be analyzed generally contains a wide variety of impurities, and there can be a case in which the efficiency of condensation in the pre-focusing column is seriously deteriorated.
For example, the use of a liquid chromatograph for separating a mixture containing a blood serum causes a problem in that proteins contained in the blood serum with a large amount are adsorbed on the column packing material and the efficiency of condensation is impaired.
In order to avoid the problem of degrading of condensation in the column packing material and to secure a high resolution separation in the separation column, it has been necessary to remove the proteins from the specimen prior to the process of liquid chromatography by conducting a preparation process.
However, such a preparation process takes a time and a substantial work load. Further, such a preparation process tends to cause a degradation in the precision of analysis. In view of above, there are provided new column packing materials that allow a direct injection of specimen containing proteins into the liquid chromatograph without conducting the process of removing the proteins. The column packing material thereby achieves the condensation of the protein-containing specimen.
These improved column packing materials use porous glass or silica gel as a carrier, and a substance having a different property is provided on the surface of the pores. By using such an improved column packing material, the large protein molecules are refused to enter the fine pores. The large protein molecules merely pass through the column without being adsorbed on the hydrophilic surface (surface outside the pores), while small molecules of drugs are adsorbed by the hydrophobic inner surface (pore inner surface).
An example of such a column packing material is disclosed in the Japanese Laid-Open Patent Publication 60-56256. In the foregoing reference, the column packing material has a protein coating on a silica surface to which an octadecyl group is bonded. The protein used for the coating may be a bovine serum albumin, and the packing material is formed by adsorption and denaturation of the protein on the octadecyl-bonded silica surface.
On the other hand, such a protein-coated ODS silica packing material has a drawback in that the adsorbed denaturation protein tends to cause an elution when used for a long time. Further, such a packing material has a problem of durability or resolution in that it is difficult to provide a column of high separation efficiency.
In order to improve the foregoing problems, it is proposed, as described in the Japanese Laid-Open Patent Publications 61-65159 and 1-123145, to provide a method of producing a column packing material according to the steps of:
(1) introducing hydrophobic groups on the inner surface and outer surface of the porous support;
(2) selectively disconnecting the hydrophobic groups from the outer surface by using an enzyme, which is a macromolecules and cannot enter the pores of the silica support; and then
(3) introducing hydrophilic groups on the outer surface.
In more detail, the Japanese Laid-Open Patent Publication 61-65159 describes a process of: bonding oligopeptide on a porous silica as a starting material to which a glycerylpropyl group is introduced, via carbonyldiimidazole; and disconnecting the phenylalanine side chain on the outer surface by a hydrolysis using carboxypeptidaze A as a proteolytic enzyme. As a result of this, glycol-phenylalanylphenyl-alanine remains on the inner surface of the column packing material as a hydrophobic ligand, while the outer surface carries a hydrophilic glycyl-glycerylpropyl group thereon.
According to the process of the Japanese Laid-Open Patent Publication 1-123145, there is described a process of: introducing a hydrophobic group on the surface of a porous silica starting material, to which an aminopropyl group is introduced, via an amide bond, by causing a reaction with octanoyl chloride under existence of triethylamine; causing a hydrolysis in the acyl group on the surface by using polymyxin acylase; and making the amino group on the outer surface to be hydrophilic by conducting a reaction with glycydol.
When the foregoing column packing material disclosed in the Japanese Laid-Open Patent Publication 61-65159 or 1-123145 is used for the pre-focusing column of a liquid chromatograph, on the other hand, there arises a problem, associated with the fact that the disclosed packing materials are formed by using an enzyme reaction, in that the production of the column packing material is complicated and the obtained column packing material tends to show a variation in the performance.
Further, there has been a problem in the conventional liquid chromatograph, which uses a pre-focusing column of large volume of 2.0 ml or more, in that the specimen supplied thereto may be diluted rather than condensed. When this occurs, the precision of detection is deteriorated inevitably. This problem becomes particularly serious in the case in which the amount of the injected specimen is very small.
It should be noted that the foregoing packing materials have a common feature of removing a substance such as proteins. In order to remove proteins selectively, the surface of the foregoing conventional packing materials is controlled hydrophobic or hydrophilic. On the other hand, such a control does not enhance the separation effect of the specimen to be analyzed.
Thus, the specimen to be analyzed is still processed by the hydrophobic surface in the foregoing conventional packing material just similarly as before, and thus, the type of the specimens, particularly the range of the specimens subjected to the pre-focusing condensation process, is essentially the same as in the conventional case.
Thus, the specimen to be condensed is limited, in the foregoing prior art, to a non-polar or low-polar substance, and the prior art cannot achieve an effective pre-focusing condensation process for ionic substances contained in a biological specimen. This problem occurs when the prior art process is applied to the analysis of a biological specimen, which may be a mixture of various substances including blood serum.
The present invention is made in view of above and has an object to provide a liquid chromatograph using a small-volume pre-focusing column which is easy to make and provides a high efficiency of condensation.
Another object of the present invention is provide a liquid chromatograph and a method of analysis, wherein a packing material having a function of cation exchange is used for the packing material of the pre-focusing column and for the packing materials of the pre-focusing column and the separation column, so that a high efficiency of resolution is achieved for the cationic substances in the specimen analyzed.
According to the invention of claim 1, there is provided a liquid chromatograph, comprising:
a separation column for separating a specimen supplied thereto together with a solvent by a pump for analysis;
flow-path control means disposed between said pump and said separation column, said flow-path control means being supplied with said solvent from said pump and said specimen from a specimen injection tube, said flow-path control means supplying said solvent to said separation column together with said specimen;
detection means supplied with said specimen separated by said separation column, said detection means analyzing said specimen;
a specimen holding mechanism holding a plurality of vessels each containing a specimen to be analyzed;
specimen injection means sampling a specimen selectively from one of said plurality of vessels, said specimen injection means supplying said sampled specimen to said flow-path control means; and
a pre-focusing column provided between said flow-path control means and said separation column, said pre-focusing column condensing said specimen that is supplied together with said solvent;
said pre-focusing column having a volume of less than 2.0 ml,
said pre-focusing column being packed with a column packing material of a porous support coated by a silicone polymer having a Sixe2x80x94Rxe2x80x94X bond (R represents a spacer part, X represents a sulfonic group) and a Sixe2x80x94Rxe2x80x2 bond (Rxe2x80x2 represents a hydrophilic group).
According to the invention of claim 2, there is provided a liquid chromatograph as claimed in claim 1, wherein the hydrophilic group Rxe2x80x2 is a hydrophilic group containing a hydroxyl group.
According to the invention of claim 3, there is provided a liquid chromatograph as claimed in claim 1, wherein the spacer part R of said column packing material is formed of a hydrocarbon residue containing 1-40 carbon atoms or a substituent of said hydrocarbon residue in which said carbon atoms are partially substituted by an oxygen atom or a nitrogen atom.
According to the invention of claim 4, there is provided a liquid chromatograph as claimed in any of claim 1, wherein said porous support constituting said packing material packed in said pre-focusing column is a silica gel.
According to the invention of claim 5, there is provided a liquid chromatograph as claimed in claim 1, wherein said separation column has a volume of less than 2.0 ml, and wherein said separation column is a cation-exchange column packed with an ion-exchange packing material having a cation-exchange function.
According to the invention of claim 6, there is provided a method of analysis, comprising the steps of:
supplying a specimen dispersed in a solvent, to a pre-focusing column, said pre-focusing column including a column body having a volume of less than 2.0 ml and filled with a porous support coated by a silicone polymer, said silicone polymer having a Sixe2x80x94Rxe2x80x94X bond (R represents a spacer part, X represents a sulfonic group) and a Sixe2x80x94Rxe2x80x2 bond (Rxe2x80x2 represents a hydrophilic group) and condensing said specimen dispersed in said solvent;
supplying said condensed specimen to a separation column for separating said specimen dispersed in said solvent; and
analyzing said separated specimen by detection means.
According to the invention of claim 7, there is provided a method of analysis, comprising the steps of:
supplying a solvent acting as a mobile phase to a flow-path control device by a pump and further supplying a specimen to said flow-path control device via a specimen injection tube connected to said flow-path control device, said flow-path control device thereby dispersing said specimen in said solvent;
supplying said specimen dispersed in said solvent to a pre-focusing column according to a pumping pressure of said pump exerted on said solvent, said pre-focusing column being packed with a packing material for condensing said specimen;
condensing said specimen by said pre-focusing column;
supplying said condensed specimen to a separation column according to a pumping pressure of said pump exerted on said solvent, said separation column separating said specimen dispersed in said solvent;
supplying said separated specimen to detection means according to a pumping pressure of said pump exerted on said solvent for analyzing said specimen,
said pre-focusing column having a volume of less than 2.0 ml,
said pre-focusing column being packed by a column packing material of a porous support coated by a silicone polymer, said silicone polymer having a Sixe2x80x94Rxe2x80x94X bond (R represents a spacer part, X represents a sulfonic group) and a Sixe2x80x94Rxe2x80x2 bond (Rxe2x80x2 is a hydrophilic group).
According to the intention of claim 8, there is provided a method as claimed in claim 7, wherein said separation column has a volume of less than 2.0 ml and wherein an ion-exchange packing material having a cation-exchange function is packed in said separation column.
According to the invention of claim 9, there is provided a method of analyzing a cationic compound in a biological specimen, by applying a method of analysis of claim 7 or 8 to said biological specimen that contains said cationic compound.
According to the invention of claim 10, there is provided a method of analyzing a biological specimen containing a cationic compound and a protein, by applying a method of analysis of claim 7 or 8 for analyzing said cationic substance in said biological specimen.
According to the invention of claim 11, there is provided a method of analyzing a biological tissue specimen acquired from any of blood plasma, blood serum, urine, saliva and a biological tissue, by applying a method of analysis as claimed in claim 7 or 8 to said biological tissue specimen as said specimen, such that a cationic substance in said specimen is analyzed.
According to the invention of claim 12, there is provided a column packing material comprising a porous support coated by a silicone polymer, said silicone polymer having a Sixe2x80x94Rxe2x80x94X bond (R represents a spacer part, X represents a sulfonic group) and a Sixe2x80x94Rxe2x80x2 bond (Rxe2x80x2 represents a hydrophilic group).
According to the invention of claim 13, there is provided a column packing material as claimed in claim 12, wherein said hydrophilic group Rxe2x80x2 of said Sixe2x80x94Rxe2x80x2 bond is a hydrophilic group having a hydroxyl group.
According to the invention of claim 14, there is provided a column packing material as claimed in claim 12, wherein said spacer part R of said Sixe2x80x94Rxe2x80x94X bond is formed of a hydrocarbon residue containing 1-40 carbon atoms or a substituent of said hydrocarbon residue in which said carbon atoms are partially substituted by oxygen atoms or nitrogen atoms.
According to the invention of claim 15, there is provided a column packing material as claimed in any of claim 12, wherein said porous support constituting said packing material is a silica gel.
According to the features of claims 1, 2 and 3, the problem of dilution of the specimen is successfully avoided by reducing the volume of the pre-focusing column of the liquid chromatograph to be less than 2.0 ml, and the condensation process proceeds reliably in the pre-focusing column. Dilution does not occur even when the amount of the specimen to be injected is small. Associated with this, the chromatograph of the present invention can provide a precise analysis.
In the present invention, the column packing material in the pre-focusing column comprises a porous support coated by a silicone polymer having a Sixe2x80x94Rxe2x80x94X bond (R represents a spacer part, X represents a sulfonic group) and a Sixe2x80x94Rxe2x80x2 bond (Rxe2x80x2 represents a hydrophilic group). In such a column packing material, a part of the outer surface thereof is hydrophilic, and thus, the problem of adsorption of proteins contained in a biological specimen does not occur even when the liquid chromatograph is used for a biological specimen. Further, the packing material is stable and provides a superior resolution, and the condensation of the specimen is achieved reliably. As no enzymes are used for the reaction, the packing material of the present invention shows an excellent reproducibility and the condensation process can be achieved with reliability.
In addition, it should be noted that the sulfonic group provided at the tip end of the spacer part R shows strong ionic interaction with cations. Thus, the column packing material has a cationic exchange function and is capable of condensing cationic compounds selectively.
Thus, the liquid chromatography of the present invention provides an excellent resolution for cationic compound specimens.
According to the feature of claim 4, a silica gel is used for the packing material packing the pre-focusing column, wherein the use of silica gel is advantageous in the point that it is free from the problem of swelling by the solvent, which is used for the mobile phase.
Thus, the liquid chromatography of the present invention provides an excellent resolution for the cationic compound specimens. Further, the reliability of analysis is improved substantially.
According to the feature of claim 5, the specimen is condensed by utilizing the cationic nature of the specimen. The specimen is then subjected to a separation process by further utilizing the cationic nature thereof.
Thus, the liquid chromatography of the present invention provides an excellent resolution for the cationic compound specimens.
According to the features of claims 6 and 7, the problem of dilution of the specimen is successfully avoided by reducing the volume of the pre-focusing column of the liquid chromatograph to be less than 2.0 ml, and the condensation process proceeds reliably in the pre-focusing column. Associated with this, the chromatograph of the present invention can provide a precise analysis.
In the present invention, the column packing material in the pre-focusing column comprises a porous support coated by a silicone polymer having a Sixe2x80x94Rxe2x80x94X bond (R represents a spacer part, X represents a sulfonic group) and a Sixe2x80x94Rxe2x80x2 bond (Rxe2x80x2 represents a hydrophilic group). In such a column packing material, a part of the outer surface thereof is hydrophilic, and thus, the problem of adsorption of proteins contained in a biological specimen does not occur even when the liquid chromatograph is used for a biological specimen. Further, the packing material is stable and provides a superior resolution, and the condensation of the specimen is achieved reliably. As no enzymes are used for the reaction, the packing material of the present invention shows an excellent reproducibility and the condensation process can be achieved with reliability.
In addition, it should be noted that the sulfonic group provided at the tip end of the spacer part R shows strong ionic interaction with cations. Thus, the column packing material has a cationic exchange function and is capable of condensing cationic compounds selectively.
Thus, the liquid chromatography of the present invention provides an excellent resolution for cationic compound specimens.
According to the feature of claim 8, the specimen is condensed by utilizing the cationic nature of the specimen. The specimen is then subjected to a separation process by further utilizing the cationic nature thereof.
Thus, the liquid chromatography of the present invention provides an excellent resolution for the cationic compound specimens.
According to the features of claims 9, 10 and 11, it becomes possible to concentrate the cationic compound contained in a biological specimen by utilizing the cationic nature of the compound.
Further, the operation of the cationic compound can be achieved by utilizing the cationic nature of the compound.
Thus, the present invention enables analysis of a biological specimen, particularly a cationic compound contained therein, with excellent resolution and separation.
According to the feature of claims 12, 13 and 14, the column packing material of the present invention has a Sixe2x80x94Rxe2x80x94X bond, with a sulfonic group bonded to a tip end of the spacer part R. As the sulfonic group exhibits a strong ionic interaction to cations, the column packing material of the present invention shows a cation-exchange function, which enables the column packing material to selectively hold or concentrate cationic compounds.
Thus, the column packing material of the present invention shows a high performance in concentrating cationic specimens.
Further, because of the presence of the Sixe2x80x94Rxe2x80x2 bond, a part of the outer surface of the column packing material of the present invention is hydrophilic in nature. Thus, the problem of adsorption of proteins is successfully avoided even when the column packing material of the present invention is used for a biological specimen that contains proteins. Further, the packing material of the present invention is stable and has an excellent resolution, and thus, it is possible to carry out the concentration with reliability by using the column packing material.
From the foregoing, it is possible to provide, according to the present invention, a stable and high-performance column packing material, which is applicable to biological specimens such as a cationic compound containing proteins.
According to the invention of claim 15, it is possible to use silica gel for the porous support of the column packing material for use in the pre-focusing column, wherein silica gel is a reliable material free from the problem of swelling, which tends to be caused by the solvent used for the mobile phase in liquid chromatography.
Thus, the present invention provided a reliable column packing material having an excellent retention performance, and hence concentration performance, for cationic specimen compounds.